1. Field of the Invention
This invention relates generally to the area of assays and in particular to the determination of homocysteine.
Homocysteine (Hcy) is one in a series of intermediates produced along the transsulfuration pathway in which methionine is eventually converted to cysteine (Cys). The exclusive source of Hcy in mammals derives from the product of the enzyme-catalyzed hydrolysis of S-adenosylhomocysteine. Once formed, Hcy may reenter the cycle through remethylation and conversion to methionine or combine with serine to form cystathionine, which is ultimately converted to Cys. The major metabolic pathway for the methylation of Hcy by methionine synthase requires vitamin B.sub.12 (cobalamine) as a methyl-transfer cofactor and 5-methyl-tetrahydrofolate as the ultimate methyl source.
Hcy and Cys are sulfhydryl amino acids that are homologs. Hcy is HOOCCH(NH.sub.2)CH.sub.2 CH.sub.2 SH and Cys is HOOCCH(NH.sub.2)CH.sub.2 SH. Hcy and Cys are immunologically related because an antibody generated against Hcy would be expected to crossreact with Cys. This is so because there is only a single carbon difference between the two compounds in the length of their amino acid side chains. Because of this immunological relationship between Hcy and Cys, it is difficult to develop an assay that can distinguish between Hcy and Cys by immunological methods.
Elevated levels of serum Hcy have been associated with insufficient intake of vitamin B.sub.12 or folate, or a deficiency in the ability to properly utilize these two vitamins. Moderately elevated levels of Hcy usually can be brought into balance by administering folate, a treatment for which there are few adverse side effects.
Both individuals who are anemic and those who are heterozygous for a defective cystathionine synthase gene are particularly susceptible to elevated Hcy levels following methionine loading. Genetic predisposition is the most common cause of moderate homocysteinuria (build-up of Hcy in urine) in otherwise healthy patients. Lastly, there appears to be a correlation between moderately elevated levels of Hcy and cardiovascular disease. For these reasons, there has been great interest in developing an accurate Hcy assay.
Several immunochemical methods for the assay of homocysteine have been described. The fundamental problem is that cysteine and homocysteine differ by only one methylene group and it is difficult to prepare antibodies that discriminate sufficiently without derivitization of one or both of the compounds. These steps add to the complexity of the method. Additionally, an immunochemical method requires that the assay be carried out on an immunochemical analyzer. Since an assay for homocysteine would be of use as a general screen in much the same way as an assay for cholesterol, HDL and LDL, it would be highly desirable to be able to run a homocysteine assay on the same instruments as the cholesterol, HDL and LDL assays.
There are several techniques to quantitate total homocysteine (Hcy) as well as distinguish between the reduced form and oxidized forms containing a disulfide, which may be free or protein-bound (primarily to albumin).
An excellent overview of the causes of homocysteinuria as well as an update on the current methods of clinical analysis can be found in Ueland, et al., Clin. Chem. 39(9):1764-1779 (1993).
An enzymatic method for a Hcy assay is described by Sundrehagen, et al., PCT/GB93/00138, where Hcy is assayed indirectly by measuring the product concentration following the enzyme catalyzed conversion of Hcy to S-adenosyl homocysteine.
High performance liquid chromatographic (HPLC) methods for Hcy and Cys are known in the art. This analytical method discriminates between Hcy and Cys by differential adsorption and elution of the compounds on a chromatographic support. Andersson, et al., Clin. Chem. 39(8):1590-1597 (1993) describe the determination of total, free and reduced Hcy and Cys.
Hcy and Cys analysis by means of a gas chromatograph-mass spectrometer is described in Allen, et al., U.S. Pat. No. 4,940,658. Allen, et al., PCT/US92/05727 describe a chromatographic assay for cystathionine, the intermediary amino acid between Hcy and Cys produced in the metabolism of methionine.
Fiskerstrand, et al., Clin. Chem. 39(2):263-271 (1993) describe a fully automated analysis of total Hcy involving fluorescent labeling of serum thiols, followed by chromatographic separation of the Hcy derivative from the other sulfur-containing compounds.
Identification of Hcy by HPLC methods often involves derivatization with fluorescent reagents such as is described in Fiskerstrand, supra, or a radioenzymatic technique such as is described in Refsum, et al., Clin. Chem. 31(4) 624-628 (1985). In addition, identification of Cys by protein sequence analysis involves derivatization with alkylating reagents. See, for example, Jue, et al., Analytical Biochemistry 210:39-44 (1993).
Unfortunately, chromatographic methods have the disadvantage of being slow and labor intensive. Furthermore, current methods of Hcy analysis require prior derivatization with fluorescent labels, such as bromobimane, in which the bromomethyl group reacts with the free thiol of Hcy, thus forming a thioether and releasing free bromide ion. The bromobimane reagent also reacts with all other free thiols in solution; therefore, chromatographic separation of the various derivatized sulfur-containing species is necessary.
There are numerous techniques for handling undesirable cross-reactants. Brynes, et al. (U.S. Pat. No. 4,952,336), describe a method of pretreating a sample with an aqueous periodate solution to eliminate cross-reactants in an amphetamine-methamphetamine immunoassay. Stevenson, PCT/GB90/01649, pertains to an improved immunoassay where the level of interference from rheumatoid factor is reduced by pretreating the sample with a reducing agent. U.S. Pat. No. 4,978,632 (Mach, et al.) pertains to an improved immunoassay where the level of interference from blood and blood products is eliminated by pretreating the sample with an oxidizing agent. These pretreatment methods only affect the cross-reactants; none of the methods affect the analyte.
As many of the current methods of Hcy analysis rely on cumbersome chromatographic techniques, there is a need for a faster and simpler assay for Hcy.
2. Description of the Related Art
U.S. Pat. No. 5,478,729 (Van Atta, et al.) discloses an immunoassay for homocysteine.
Simons, et al., (Simons 1) J. Am. Chem. Soc., (1976) 98(22), 7098-7099, disclose the reaction of amino acids with o-phthalaldehyde and thiols to form fluorescent products.
Simons, et al., (Simons 2), Anal. Biochem. (1978) 90(2), 705-725, disclose the fluorescent properties of 1-alkyl (and aryl) thio-2-alkylisoindoles formed by the reaction of o-phthalaldehyde and thiols with primary amines.
Simons, et al., (Simons 3), J. Org. Chem. (1978) 43(14), 2886-91, disclose the formation of 1-alkylthio-2-alkylisoindoles from o-phthalaldehyde, mercaptols and amino acids.
Alvarez-Coque, et al., Spectrochim. Acta, Part A (1988) 44A(12), 1461-4, disclose the reaction of cysteine with o-phthalaldehyde to produce a fluorescent compound at elevated temperatures but not at room temperature.
Chen, et al., Biochim. Biophys. Acta (1979) 576(2), 440-55, disclose the fluorescence quantum yields of the product of o-phthalaldehyde, amino acids and thiol compounds and show that cysteine reacts to give an unstable, weakly fluorescent product unless its sulfhydryl group is blocked.
Lee, et al., J. Biol. Chem., (1979) 254, 6248, disclose the modification of the cysteine mercapto group to facilitate formation of a highly fluorescent product upon reaction with o-phthalaldehyde.
Metz, et al., J. Chromatogr. (1985) 330(2) 307-13, disclose the post-column derivatization of cysteine and other .alpha.-aminothiols with phthalaldehyde and determination of the structures of the fluorescent products.
Puri, et. al., Anal. Bioochem. (1988) 173(l), 26-32, disclose monitoring of the reaction of glutathione, homocysteine and cysteine with o-phthalaldehyde by measuring the fluorescence of the isoindole derivatives that are formed to compute molar transition energies.
Simons (Simon 4) , J. Chem Soc., Chem Comm., (1977) 11, 374, disclose the formation of 1-t-butylthio-2-propylisoindole from o-phthalaldehyde, t-butylthiol and propylamine.
Simpson, et al., (Simpson 1) J. Chromatogr. (1983) 261(3), 407-14, disclose off-line liquid chromatographic-mass spectrometric studies showing that the structure of the fluorescent products of the reaction of o-phthalaldehyde, primary amines and thiols are 1-alkylthio-2-alkyl-substituted isoindoles.
Simons, et al., (Simons 5) J. Org. Chem. (1981) 46(23), 4739-4744, disclose the formation of dark red substituted isoindoles upon reaction of dimethyl acetylenedicarboxylate with 1-alkylthio-2-propylisoindoles.